Industrial Crops and Products 87 (2016) 64–70

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Sacha inchi (Plukenetia volubilis L.) oil composition varies with

changes in temperature and pressure in subcritical extraction with
n-propane
Ana Beatriz Zanqui a , Claudia Marques da Silva a , Damila Rodrigues de Morais c ,
Jandyson Machado Santos c , Suellen Andressa Oenning Ribeiro a ,
Marcos Nogueira Eberlin c , Lúcio Cardozo-Filho b , Jesuí Vergílio Visentainer a ,
Sandra Terezinha Marques Gomes a , Makoto Matsushita a,∗
a
Department of Chemistry, State University of Maringá, UEM, Maringá, PR 87020-900, Brazil
b
Department of Chemistry Engineering, State University of Maringá, UEM, Maringá, PR 87020-900, Brazil
c
ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of Campinas—UNICAMP, Campinas, SP 13083-970, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:
Received 22 January 2016
Received in revised form 6 April 2016
Accepted 7 April 2016
Chemical compounds studied in this article:
Stigmasterol (PubChem CID: 5280794)
Sitosterol (PubChem CID: 222284)
Cholestane (PubChem CID: 6857534)
␣-Linolenic acid (PubChem CID: 5280934)
Linoleic acid (PubChem CID: 5280450)
Oleic acid (PubChem CID: 445639)
Palmitic acid (PubChem CID: 985)
Stearic acid (PubChem CID: 5281)
Vaccenic acid (PubChem CID: 5281127)
Sacha inchi is an oilseed crop that shows high oil content and it is an excellent source of polyunsaturated
fatty acids. Temperature and pressure can influence in lipid composition on the subcritical extraction
with n-propane. The highest extraction yield obtained was 30% under the conditions of 60 ◦ C and 12 MPa.
The extracted oil presented 442 mg of fatty acid g−1 in terms of alpha linolenic acid. Analysis by easy
ambient sonic-spray ionization mass spectrometry showed a typical TAG (Triacylglycerols) profile for
Sacha inchi oil with major ions of m/z (mass-to-charge ratio) 895 (ALA-ALA-ALA), 897 (ALA-ALA-LA), 899
(ALA-LA-LA) and 901 (LA-LA-LA or OL-LA-ALA) in a form of adduct of [TAG + Na]+ , in which ALA is alfa
linoleic acid, LA is linoleic acid and OL is oleic acid. The phytosterols content (86.39–101.92 mg 100 g−1
of total lipids) varied according to the conditions applied for the n-propane extraction. In general, the
subcritical fluid extraction with n-propane was found to preserve the important Sacha inchi oil bioactive
constituents, that is, fatty acids, triacylglycerols and phytosterols. Regression analysis also showed that
higher temperatures and pressures result in higher yields of phytosterols.
© 2016 Elsevier B.V. All rights reserved.

Keywords:
Plukenetia volubilis L.
Subcritical fluid extraction
Alpha linoleic acid
Easy ambient sonic-spray ionization
Omega 3
Phytosterols

1. Introduction Romero et al., 2009). The oil obtained from P. volubilis L. is an

Sacha inchi (Plukenetia volubilis L.) is an oilseed crop mainly
originated from the Amazon Rainforest in Peru. This crop shows a
high oil content which varies from 40 to 60% in weight (Follegatti-
Abbreviations: ALA, ␣-linolenic acid; FA, fatty acids; HDL, high density lipopro-
tein; LA, linoleic acid; MUFA, monounsaturated fatty acids; OL, oleic acid; PCA,
principal components analysis; PUFA, polyunsaturated fatty acids; S, stearic acid;
SE, soxhlet method; SFA, saturated fatty acids; SubFE, subcritical fluid extraction
method; TAG, triacylglycerols; TL, total lipids.
∗ Corresponding author.
E-mail address: mmakoto@uem.br (M. Matsushita).
excellent source of polyunsaturated fatty acids (PUFA) such as ALA
(18:3n-3, ␣-linolenic acid) and LA (18:2n-6, linoleic acid), which are
precursors of the omega-3 (n-3) and omega-6 (n-6) series, respec-
tively. Since our metabolism is incapable to synthesize both LA and
ALA, they are also essential fatty acids (FA) which should be present
in the human diet. In the last years, these FA have also received
significant emphasis since their health benefits have been firmly
confirmed (Gutiérrez et al., 2011; Maurer et al., 2012; Souza et al.,
2013). According to Chirinos et al. (2013), Sacha inchi grain is an
excellent source of bioactive molecules such as phytosterols which
decrease the risk of certain types of cancer and reduce cholesterol
in the blood (Lagarda et al., 2006; Moreau et al., 2002).

http://dx.doi.org/10.1016/j.indcrop.2016.04.029
0926-6690/© 2016 Elsevier B.V. All rights reserved.
 A.B. Zanqui et al. / Industrial Crops and Products 87 (2016) 64–70 65

The ␣-linolenic acid is also known to prevent cardiovascular dis-

eases, decrease the risk of heart and other chronic diseases, increase
HDL (high density lipoprotein) blood quantities, prevent cancer
and relief the effects of autoimmune diseases, rheumatoid arthri-
tis and depression (Calder, 2006; Capurso et al., 2014; Jump, 2002;
Ruxton et al., 2005; Scoditti et al., 2014; Simopoulos, 2011). Oleic
acid (18:1n-9, OL) is not an essential FA to humans but its consump-
tion has also been associated with numerous healthy benefits such
as antithrombotic effect (Capurso et al., 2014; Scoditti et al., 2014).
To extract triacylglycerols (TAG) from seeds, the food industry
has mainly used extractions by hexane or others organic solvents
such as petroleum ether, ethylic ether, methanol and chloroform.
However, the use of hexane for food processing is facing restric-
tions, due to the large amounts of toxic solvents normally required
as well as the need of demanding operations for solvent removal.
The presence of solvent residues in the final product is also a con-
cern. Therefore, studies have been performed to develop alternative
Fig. 1. Scheme of the experimental unit for lipid extraction by subcritical
methods of lipids extraction. fluid, where: A—n-propane cylinder; B—pump-type syringe; C—puller; D and
Supercritical CO2 extraction is very beneficial for oil process- E—thermostated baths; F—micrometric valve; G—expansion valve; H—oil outlet;
ing, particularly when dealing with functional food, for which the I—bottle collector; J—thermoregulator.
demand for “natural” products obtained through clean solvent-free
technology has increased significantly in recent years (Martínez Table 1
and Aguiar, 2014). Carbon dioxide supercritical fluid oil extraction Factors and levels evaluated in the experimental design full 22 with central point
for the subcritical fluid extraction.
results in nearly pure oils and relative high yields but is quite time-
consuming. Subcritical oil extraction using propane is faster as Factors/Test Temperature (◦ C) Pressure (MPa) Density (g mL−1 )
compared to CO2 due to the higher solubility of lipids in n-propane. A 45 10 0.489
n-propane has also been reported as an efficient and non-toxic oil B 30 8 0.504
solvent. But the purity and yields of n-propane subcritical extrac- C 30 12 0.513
tion as well as the final composition of the oil has been found to D 60 8 0.460
E 60 12 0.474
considerably vary depending on the extraction conditions (Silva
et al., 2015; Follegatti-Romero et al., 2009; Nimet et al., 2011;
Pederssetti et al., 2011; Zanqui et al., 2015a,b).
temperature-controlled thermostatic bath at 10 ◦ C (Nimet et al.,
The aim of this study was evaluate the influence of pressure
2011).
and temperature in the composition of the Sacha inchi oil (fatty
The extraction was carried out with 1 mL min−1 of n-propane
acids, phytosterols and TAG) obtained via subcritical extraction
flow during 60 min, controlled by an expansion valve (Autoclave
with n-propane. The oils obtained either by n-propane subcritical
Engineers) maintained at 80 ◦ C using a thermoregulator (Tholz,
extraction or the conventional Soxhlet method was also compared
model CTM-04E).
via analysis performed by gas chromatography with flame ioniza-
The data analysis was performed according to the 22 factorial
tion detection (GC-FID), GC coupled to mass spectrometry (GC–MS)
design (Table 1), with three replications of the central point and
and easy ambient sonic-spray ionization coupled to mass spec-
the responses were the extraction yield, quantity of some fatty
trometry (EASI-MS).
acids and phytosterols. In Table 1 is also reported the densities of
n-propane in its conditions of temperature and pressure (NIST web
Book).
2. Material and methods

2.1. Sample preparation 2.3. Soxhlet extraction method (SE)

Three kilograms (3 kg) of Sacha inchi (P. volubilis L.) obtained
from regional market of Bogota (Colombia) were used as repre-
sentative samples. The Sacha peanut peel and grain fractions were
carefully separated. The grains were ground in a Wiley mill (Tecnal,
model TE 631/3, Brazil) to obtain a fine flour that was then sieved,
using the fraction that passed through a 6 mesh and not that passed
through a 9 mesh Tyler series sieve (WS Tyler, USA). Later, the sam-
ple was thoroughly mixed and vacuum packed in polyethylene bags
and frozen at −18 ◦ C.
2.2. Subcritical fluid extraction method
For lipid extraction with pressurized n-propane in a labora-
tory scale (Fig. 1), 15.0 g of Sacha inchi and porcelain beads were
introduced into the extractor with an internal volume of 53.4 cm3 .
The porcelain beads were inserted so that the whole volume of
the extractor was filled. The solvent n-propane (White Martins,
99.5% purity) was pressurized via a pump-type syringe with a
Approximately 4.0 g of Sacha inchi were extracted in a Soxhlet
extractor (Nova Etica, Brazil) using a mixture of ethyl ether-
petroleum ether (1:1 v−1 ) for 16 h at boiling point. Later, the solvent
was evaporated with aid heated water at 65 ◦ C and total lipids con-
tent was determined gravimetrically (Soxhlet, 1879).
2.4. Fatty acid composition
Fatty acid methyl esters (FAME) were prepared by the methy-
lation of total lipid (TL) according to Hartman and Lago (1973)
and analyses were performed in triplicate. Methyl esters were
separated by GC-FID (Trace Ultra 3300 model-Thermo Scientific)
equipped with a flame ionization detector and a cyanopropyl cap-
illary column (100 m × 0.25 i.d., 0.25 ␮m film thickness, CP-7420
Varian, EUA). The injector, detector and gases conditions, and
the main operational parameters were according to Zanqui et al.
(2015b). The peak areas were determined by the ChromQuest 5.0
software. For FA identification, retention times were compared
with those of standard methyl esters. Quantification (in mg FA g−1
66 A.B. Zanqui et al. / Industrial Crops and Products 87 (2016) 64–70

Fig. 2. EASI(+)-MS (left) and expanded EASI(+)-MS in the m/z 800–1000 TAG ions range (right) for Sacha inchi oils obtained by n-propane subcritical extraction in different
conditions (A–E) or via Soxhlet extraction (F).

of TL) was performed against tricosanoic acid methyl ester as an analyses were performed in triplicate. Data were acquired and pro-
internal standard (23:0) (Joseph and Ackman, 1992). cessed using the LCMS solution v.3.70 software (Shimadzu, Japan).

2.6. Phytosterols and tocopherols composition

2.5. Triacylglycerol composition
The EASI-MS fingerprintings of the Sacha inchi oil were obtained
using a single quadrupole mass spectrometer LCMS-2010 EV (Shi-
madzu, Japan) with a home-made EASI source (Haddad et al., 2008;
Haddad et al., 2006) operating in the positive ion mode—EASI(+).
The EASI(+) source was operated with a methanol flow rate of
20 ␮L min−1 and 2 L min−1 for the nebulizing gas (N2 ). The surface-
entrance angle was fixed in 30◦ . A single droplet of the Sacha inchi
oil (1 ␮L) was deposited on a paper surface (brown Kraft envelope
paper) and EASI(+)-MS data were collected for over 30 s, scan-
ning over the m/z (mass-to-charge ratio) 100–1200 range. The data
Phytosterols and tocopherols were simultaneously evaluated by
GC–MS (Du and Ahn, 2002). The extracted oils were previously
derivatized (Beveridge et al., 2002), using N-O-bis (trimethylsilyl)
trifluoracetamide (BSTFA) (Sigma-Aldrich Co., Brazil) as the deriva-
tizing reagent. The analysis were performed using a GC–MS Agilent
7890A (Agilent-Technologies, USA) using a DB-5 capillary column
(5% phenil, 95% methilpolisiloxane, 30 m × 0.25 mm × 0.25 ␮m) (J
& W Scientific). The MS was an Agilent 5975C instrument (VL MSD)
and used 70 eV EI and a quadrupole mass analyzer. Data analysis
used database NIST MS Search version 2.0 2014. The GC–MS were
those previously described by Zanqui et al. (2015a). Quantitation
 A.B. Zanqui et al. / Industrial Crops and Products 87 (2016) 64–70 67

Table 2 sizes ranging from 3 to 6 mesh, and an oil yield of only 18.8% was
Yields of total lipids extracted by subcritical and Soxhlet extraction.
obtained. Therefore was decided to reduce the size of the particles
Test Temperature (◦ C) Pressure (MPa) Extraction Yield (%) to a range of 6–9 mesh, obtaining statistically different yields for
A 45 10 29.2b ± 0.5 the Soxhlet and SubFE extractions. That is, the extraction yield for
B 30 8 28.0b ± 0.5 the SubFE method was 30% lower than that for SE extraction.
C 30 12 26.7b ± 0.5
D 60 8 28.9b ± 0.5 3.2. Composition
E 60 12 29.7b ± 0.5
SE – – 42.0a ± 1.0
Table 3 shows the FA compositions of the SubFE and SE oils.
Mean values. Means followed by different letters in the same column demonstrated
The predominant FA in the lipid composition of Sacha inchi oil
significant difference by Tukey’s test (p < 0.05). A–E: letters representing the testing
of extraction with subcritical fluid. SE = Soxhlet method. (442 mg g−1 ) was found to be the alpha-linolenic acid (18:3n-3).
This%FA closely matches that obtained by Maurer et al. (2012).
Souza et al. (2013) also reported approximately 438.77 mg g−1 of
was performed using 5␣-cholestane (Sigma-Aldrich Co., Brazil) (Li
this FA in the oil. The same lipids as those reported in Table 3
et al., 2007) as the internal standard.
were extracted with solvents and by SubFE extraction, which shows
that these extraction methods provide similar FA compositions.
2.7. Statistical analysis
Follegatti-Romero et al. (2009) also studied the lipid composition
of the Sacha inchi oil extracted by supercritical CO2 , and found a
Fatty acids, tocopherols and phytosterols analyses were per-
composition similar to that reported in Table 3. This similarity indi-
formed in triplicate. Means and standard deviations of the
cates that n-propane also extracts the same FAs as those extracted
analytical error propagation were calculated and the results were
by the conventional SE methodology. Statistical analysis indicates
submitted to Tukey’s test at 5% probability, using the Statistica
significant differences in FA levels for the SubFE and SE methods
software, version 8.0 (StatSoft, 2007). Data was also submitted to
(Tables 3 and 4).
principal component analysis (PCA).
In relation to the summations and n-3/n-6 ratios of the extracted
Analysis of variance was used to evaluate the effect of inde-
Sacha inchi oil (Table 4), no significant differences in total saturated
pendent variables on the responses using mathematical model
(SFA) FA, monounsaturated (MUFA) and polyunsaturated (PUFA) n-
expressed by equation 1 (Ri = response, ␤0 = constant, and ␤1, ˇ2
3 and n-6 were observed using both methods. A relatively higher
and ␤12 = regression terms) where T refers to temperature, P refers
concentration of SFA and lower concentration of PUFAs in SE lipid
to pressure, using the Design Expert software, version 7.1.3 (Stat-
composition were however observed. The level of saturated FA was
Ease, 2008).
33% higher for the SE oil as compared to that using SubFE, showing
Ri = ˇ0 + ˇ1 T + ˇ2 P + ˇ12 TP (1) a bias against saturated FA for the SubFE method.
The n-3/n-6 ratio was 1.32 for TL. According to the World Health
3. Results and discussions Organization (WHO, 1995), balanced ingestion of omega-3 and 6
series FAs are recommended since these compounds act in chronic
3.1. Subcritical fluid extraction method (SubFE) and soxhlet diseases and in cardiovascular and hypertension prevention.
method (SE)
3.3. Typification of Sacha inchi oil via EASI-MS fingerprinting
Table 2 shows the yields for both the SubFE with n-propane
and SE using different temperatures and pressures. For the SubFE The EASI-MS technique has been widely applied for vegetable
extraction, preliminary tests were made with larger particles, with oil typification and quality control (Bataglion et al., 2015; Simas

Table 3
Quantification of fatty acids of Sacha inchi oil.

FA (mg g−1 of TL) A B C D E SE

16:0 40.05b ± 0.6 40.28b ± 0.6 40.19b ± 0.2 40.04b ± 0.3 39.73b ± 0.6 56.66a ± 1.0
18:0 28.43b ± 1.8 27.80b ± 1.4 27.95b ± 0.7 27.46b ± 1.0 28.83b ± 1.1 34.63a ± 0.6
18:1n-9 c 81.40a ± 2.2 80.82a ± 1.2 80.57a ± 0.2 81.22a ± 1.4 81.11a ± 1.4 82.38a ± 1.9
18:1n-7 4.76a ± 0.2 4.64a ± 0.3 4.76a ± 0.1 4.75a ± 0.1 4.72a ± 0.1 4.92a ± 0.1
18:2n-6 336.38a ± 0.9 335.74a ± 1.0 335.51a ± 0.6 334.22a ± 2.2 334.23a ± 1.0 325.08b ± 0.9
18:3n-3 440.04a ± 4.9 443.44a ± 2.9 443.29a ± 1.7 443.06a ± 2.1 441.28a ± 4.1 425.30b ± 2.0

Mean values ± standard deviation; Means followed by different letters in the same line demonstrated significant difference by Tukey’s test (p < 0.05). A–E: letters representing
the testing of extraction with subcritical fluid, where A = 45 ◦ C/10 MPa; B = 30 ◦ C/8 MPa; C = 30 ◦ C/12 MPa; D = 60 ◦ C/8 MPa; E = 60 ◦ C/12 MPa; SE = Soxhlet method. Detection
limit = 0.015 mg g−1 .

Table 4
Summations and n-3/n-6 ratio of Sacha inchi oil extracted by subcritical fluid method using n-propane.

Summations (mg g−1 of TL) A B C D E SE

SFA 68.48b ± 1.9 68.09b ± 1.6 68.14b ± 0.7 67.51b ± 1.0 68.56b ± 1.3 91.30a ± 1.2
MUFA 86.16a ± 2.2 85.46a ± 1.3 85.33a ± 0.2 85.97a ± 1.4 85.82a ± 1.4 87.31a ± 2.0
PUFA 776.42a ± 4.9 779.19a ± 3.0 778.81a ± 1.8 777.28a ± 3.0 775.52a ± 4.2 750.38b ± 2.2
n-3 440.04a ± 4.9 443.44a ± 2.9 443.30a ± 1.7 443.06a ± 2.2 441.28a ± 4.1 425.30b ± 1.9
n-6 336.38a ± 0.9 335.74a ± 1.0 335.51a ± 0.6 334.22a ± 2.2 334.23a ± 1.0 325.08b ± 0.9
Ratio A B C D E SE
n-3/n-6 1.32a ± 0.1 1.32a ± 0.1 1.32a ± 0.1 1.33a ± 0.1 1.32a ± 0.1 1.31a ± 0.1

Mean values ± standard deviation; Means followed by different letters in the same line demonstrated significant difference by Tukey’s test (p < 0.05). A–E: letters representing
the testing of extraction with subcritical fluid, where A = 45 ◦ C/10 MPa; B = 30 ◦ C/8 MPa; C = 30 ◦ C/12 MPa; D = 60 ◦ C/8 MPa; E = 60 ◦ C/12 MPa; SE = Soxhlet method. SFA = total
saturated fatty acids; MUFA = total monounsaturated fatty acids; PUFA = total polyunsaturated fatty acids; n–6 = total n-6 fatty acids; n–3 = total n-3 fatty acids.
68 A.B. Zanqui et al. / Industrial Crops and Products 87 (2016) 64–70

Table 5
Attribution of ions detected by EASI(+)-MS for the Sacha inchi oil.

FA Composition [DAG + Na]+ m/z [DAG + K]+ m/z [Monohydroperoxide + Na]+ m/z Monohydroperoxide + K]+ m/z

LA-LA 639 655

TAG Composition [TAG + Na]+ m/z [TAG + K]+ m/z [Monohydroperoxide + Na]+ m/z Monohydroperoxide + K]+ m/z
LA-LA-P 875 901
LA-LA-P/ALA-OL-P 877 903
ALA-ALA-ALA 895 911 927 943
LA-ALA-ALA 897 913 929 945
LA-LA-ALA 899 915 931 947
LA-LA-LA/OL-LA-ALA 901 917 933 949
LA-LA-OL 903 919
LA-LA-S 905 921

P = Palmitic Acid; S = Stearic Acid; OL = Oleic Acid; LA = Linoleic Acid; ALA = Linolenic Acid.

Table 6
Quantification of phytosterols and tocopherols in Sacha inchi oil extracted by subcritical fluid method using n-propane (mg 100 g−1 of TL).

Point (␥ + ␦) Tocopherols Stigmasterol Sitosterol Sum of phytosterols

A 104.88a ± 2.4 36.27b ± 0.6 50.13b ± 0.1 86.39b ± 0.7

B 105.44a ± 0.3 36.11b ± 0.5 50.67b ± 0.1 86.78b ± 0.6
C 102.58a ± 0.1 34.61b ± 1.8 43.46c ± 1.9 78.07c ± 2.3
D 104.32a ± 4.6 45.63a ± 0.2 56.29a ± 0.7 101.92a ± 0.8
E 104.46a ± 3.6 41.87a ± 2.8 50.11b ± 2.7 91.98b ± 3.3

Mean values ± standard deviation; Means followed by different letters in the same column demonstrated significant difference by Tukey’s test (p < 0.05). A–E: letters
representing the testing of extraction with subcritical fluid, where A = 45 ◦ C/10 MPa; B = 30 ◦ C/8 MPa; C = 30 ◦ C/12 MPa; D = 60 ◦ C/8 MPa; E = 60 ◦ C/12 MPa.

et al., 2010; Zanqui et al., 2015a) due the various advantages of this Table 7
Analysis of variance for the response sum of phytosterols of Sacha inchi oil extracted
ambient MS method. It provides undisturbed sampling and high-
by SubFE method.
speed of data acquisition, ease of analysis, low cost, direct analysis,
voltage-free ionization and no need for pre-separation (Alberici Source Sum of Degrees of Mean F value p-value
squares freedom square
et al., 2010). Major oil components are identified via the specific
m/z values of typically [M + Na]+ ions (Cabral et al., 2013; Haddad Model 888.39 3 296.13 47.01 <0.0001
et al., 2006). T 440.77 1 440.77 69.98 <0.0001
P 34.89 1 34.89 5.54 0.0404
Fig. 2 shows the EASI(+)-MS data of the Sacha inchi oil obtained
T.P 412.73 1 412.73 65.53 <0.0001
either by the SE and SubFE in different conditions. Note that indeed Curvature 32.62 1 32.62 5.18 0.0461
unique and contrasting triacylglycerols (TAG) profiles are obtained Pure Error 62.99 10 6.30 – –
for both extraction methods. TAG ions were detected mainly as Total 984.00 14 – – –
adduct of [TAG + Na]+ , but minor as adduct of [TAG + K]+ series T = temperature; P = pressure.
separated by 16 m/z units was also observed. The TAG ions were
detected along the ca. m/z 800–950 range (Fig. 2) whereas oxidized
TAG ions appear on the ca. m/z 940–970 range. (ALA-ALA-LA), 899 (ALA-LA-LA) and 901 (LA-LA–LA or OL-LA-ALA)
But oils obtained either by n-propane SubFE in different condi- (Fig. 2). The authors also used HPLC to monitor hydrolysis and oxi-
tions or by SE showed nearly identical EASI(+) spectra (Fig. 2). This dation of Sacha inchi oils using prior selective solid phase extraction
similarity demonstrates both the reliability of EASI(+)-MS as a typ- (SPE). Note therefore that the same information can be obtained by
ification protocol for such oils regardless the extraction protocol as EASI(+)-MS in seconds without any sample preparation at all since
well as that very similar TAG compositions are also obtained regard- [DAG + Na]+ (diacylglycerol) ions are detected in the m/z 400–700
less the temperature and pressure used. The EASI(+)-MS data for range, as example the ion of m/z 639 with two chains from Linoleic
the SE (Fig. 2-F) also showed a set of unique ions mainly separated acid (LA-LA). For oxidation, hydroperoxide TAG ions were detected
by 44 Da detected along the m/z 400–800 range, but these ions are in the m/z 929 to m/z 947 range. Note also that both the DAG (dia-
probably from polyethyleneglycol (PEG) present as contamination cylglycerols) and hydroperoxide TAG ions were detected in quite
from the SE method. low abundances indicative an extracted oil of high quality.
Table 5 shows the ions assignments from Fig. 2. Note that, for- Interestingly, when compared to other Amazon oils such as Açai,
tunately, the FA composition from the GC-FID analysis (Table 3) Buriti (Simas et al., 2010) and Andiroba oils (Cabral et al., 2013), the
closely matches the FA composition inferred from the intact TAG extracted Sacha inchi oil investigated herein show extraordinarily
composition revealed by EASI(+)-MS (Table 5), which was obtained much high contents of bioactive FA such as ALA and LA with great
in a much simpler and faster way. For instance, LA and ALA are predominance of TAG from the combination of theses acids. Indeed
determined to be predominant by both techniques. the faster and simpler EASI(+)-MS technique was able to display
Chasquibol et al. (2014) used RP-HPLC to characterize Sacha profiles of oil composition in regard to TAG, DAG and oxidized TAG
inchi oil extracted by hydraulic pressing and found the same pre- quite similar to both GC-FID and HPLC data.
dominant TAG, that is, mainly those composed by three chains
from linolenic acid (ALA), two chains from linolenic acid (ALA)
and one of Linoleic acid (LA), one chain from linolenic acid (ALA) 3.4. Phytosterols and tocopherols composition
and two of Linoleic acid (LA), and three chains from linoleic acid
(LA), respectively for the TAG ALA-ALA-ALA, ALA-ALA-LA, ALA-LA- Table 6 shows tocopherols and phytosterols composition in
LA and LA-LA-LA. A similar profile was detected by the EASI(+)-MS Sacha inchi lipids extracted by the SubFE method. There was no
of Fig. 2, via the [TAG + Na]+ ions of m/z m/z 895 (ALA-ALA-ALA), 897 significant difference on tocopherols levels. Point D stood out with
higher values for all phytosterols.
 A.B. Zanqui et al. / Industrial Crops and Products 87 (2016) 64–70
Fig. 3. Response surface for the sums of phytosterols of Sacha inchi oil extracted by
subcritical fluid extraction with n-propane.
Chirinos et al. (2013) studied 16 different cultivars of Sacha
inchi and reported 21.2–26.9 mg 100 g−1 of stigmasterol extracted
by the SE method. They also found 45.2–53.2 mg 100 g−1 of sitos-
terol. These values are similar to those obtained herein (Table 6),
but the sum of phytosterols shows that our SubFE protocol was
able to extract 25% more phytosterols than that used by Chirinos
et al. (2013). They also reported tocopherol levels, and the sum of
tocopherols was close to those obtained herein.
3.5. Statistics and multivariate analyses
Table 7 shows the results obtained by ANOVA for the responses
of phytosterols of Sacha inchi oil extracted by the SubFE method.
The ANOVA for the sum of phytosterols (Ri ) of Sacha inchi oil
extracted by SubFE process showed that both temperature and
pressure factors were significant for the model. The analysis of
the effects indicated that the temperature and the temperature x
pressure interaction effect were the most significant factors of the
Fig. 4. PCA of sums de FA for Sacha inchi oil. A–E: letters representing the testing of extraction with subcritical fluid. SE = Soxhlet extraction method. MUFA = total monoun-
saturated fatty acids; PUFA = total polyunsaturated fatty acids; n–3 = total n-3 fatty acids; n–6 = total n-6 fatty acids.
69
extraction process with contributions of 44.8 and 42.0%, respec-
tively.
Via Eq. (2), it was possible to conclude that all studied factors
contribute to the increase of the extraction of phytosterols in Sacha
inchi oil, and the greatest yield was obtained at 60 ◦ C and 12 MPa.
The ANOVA also indicates that the model was significant and con-
firms that factors such as temperature and pressure in the SubFE
with n-propane may influence selectively in the lipophilic bioactive
composition of Sacha inchi oil (Fig. 3).
Ri = 88.29 + 6.06T +1.71P + 5.86TP (2)
R2 = 0.934
Principal components analysis (PCA) (Fig. 4) was applied to find
correlations for the FA summations in relation to both SE and SubFE.
PC1 (Principal component 1) and PC2 (Principal component 2)
accounted for 46.89% and 11.99% of data variance, respectively. The
remaining PCs yielded progressively smaller eigenvalues (P < 1),
therefore PC1 and PC2 were indeed the PCs that better describe the
data. In the PC1 x PC2 plot, two groups related to both extraction
methods were formed, showing that they provided significantly
different FA compositions for the Sacha inchi oils. The extraction
with subcritical fluid approximated the maximum values of n-6,
n-3 and PUFA, showing a good correlation with these summations.
The solvent extraction method showed the highest composition of
SFA and MUFA. So PCA confirmed that the Sacha oil extracted using
n-propane as a subcritical fluid is a selective method.
4. Conclusions
In subcritical extraction using n-propane, the reduction of par-
ticle size contributed to higher yields of lipids. The extraction yield
obtained by SubFe method was about 28%. As rapidly and easily
determined by EASI(+)-MS directly from the undisturbed sample,
and also via comparison with GC-FID results, the subcritical fluid
extraction with n-propane can provide a Sacha inchi oil with a
rich composition of bioactive compounds (fatty acid, triacylglyc-
erol, phytosterols and tocopherols). The temperature and pressure
70 A.B. Zanqui et al. / Industrial Crops and Products 87 (2016) 64–70

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